The Vinca Alakloids, including vinvblastine and vincristine, isolated from the plant Vinca rosea L., are important chemotherapeutic agents which have been found to have clinical activity against a spectrum of human cancers. It is generally assumed that the mechanism for their cytotoxic, anitmitotic, and antineoplastic activity is related to their binding to the tubulin dimer of microtubules, with the subsequent depolymerization and disruption of the cellular microtubular network including the mitotic spindle. However, evidence from several studies now suggests that binding to mitotic spindle tubulin dimers cannot account for their pronounced cytotoxic effects on slow proliferating, sensitive cells and interphase cells, since in these cells cytotoxicity is evident long before mitotic arrest is manifest.
Tubulin also exists as a plasma membrane component in brain and thyroid tissues, and in vinblastine-sensitive human lymphoblasts with leukemic origin. The possibility that these drugs may exert their cytotoxic effects by interacting with the membrane tubulin component rather than cytoplasmic microtubules suggests that the mechanism for cytotoxic and cytostatic effects of these alkaloids may be more complex than simple depolymerization and disruption of mitotic spindles.
For example, Vinca alkaloids have been shown to inhibit the incorporation of .sup.3 H-uridine into RNA and .sup.3 H-thymidine in DNA. These effects may be due to the specific inhibition of nucleic acid synthesis as well as inhibition of nucleotide uptake into the cells. Similarly, vinblastine can precipitate a number of acidic proteins and nucleic acids in addition to tubulin.
Unfortunately, our knowledge of the mechanism involved in expression of Vinca alkaloid resistance in cancer cells is limited. While oversynthesis of particular proteins in Vinca alkaloid resistance cells has been noted, no attempt has been made to discover relationships between such proteins and their possible interactions with these drugs.
Previous studies of drug-protein interactions required purified protein components for equilibrium binding measurements under optimum conditions. Accordingly, these studies were costly and difficult to perform.
Affinity labelling of proteins with photoactive ligands is a powerful tool for probing biochemical targets. In particular, photoaffinity labelling has been used for identification, purification, and characterization of mediators of biological, physiological, and pharmacological activities. The photoaffinity labelling technique allows for investigation of drug-protein interactions in order to identify an acceptor molecule in a mixture of candidates or to identify a specific component of a multi-subunit system. During photoaffinity, a reversible complex presumably forms between the photoactive analog and unique acceptor sites of specific polypeptides which preferentially recognize the characteristic structure of the drug. Upon irradiation with UV light, the analog is converted into a highly reactive nitrene intermediate which will covalently interact with the acceptor sites. A particular functional group at the acceptor site need not be present because the photogenated species can react even with carbon-hydrogen bonds.
The exposure of malignant cell lines to natural product cytotoxic drugs such as vinblastine, actinomycin D, adriamycin, or colchicine, frequently results in the isolation of populations of cells with resistance to the selecting agent as well as a collateral resistance to other mechanistically distinct and structurally unrelated compounds. The mechanisms by which these cell lines become multidrug-resistant is unknown, but it is thought to be related to a parallel reduction in the cellular accumulation of those drugs to which the cells are resistant. The multidrug-resistant phenotype also is characterized by the presence of a 150-180 kDa surface membrane glycoprotein (gp150-180) which occurs in multidrug-resistant cells in direct proportion to the degree of their acquired drug-resistance. The relationship of gp150-180 to multidrug resistance is not known. It may accumulate only as a secondary consequence of the multidrug-resistant phenotype. Alternatively, gp150-180 could promote multidrug-resistance by direct or indirect effects on membrane permeability drug transport, or drug binding.